Multiplex polymerase chain reaction-restriction fragment length polymorphism assay discriminates of rabbit, rat and squirrel meat in frankfurter products

Abstract The demands for rabbit meat are rapidly growing and Rabbitry is becoming a mean of livelihood for many youths. Rats and squirrels are very close relatives of rabbits, could be hunted freely or raised in domestic farming and so could be substituted in expensive rabbit meat. This study, for the first time, developed and validated a tetraplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay to identify and discriminate rabbit, rat and squirrel meat under raw and processed foods. Four sets of primes amplified 123, 108, 243, and 141 bp fragments from rabbit, rat, squirrel and all eukaryotes, respectively. Specificity was confirmed through sequencing and RFLP analysis. When PCR products were digested with Bts I Mut I and BtsC I enzymes, distinctive fingerprints (115 & 8 bp for rabbit; 64 & 44 bp for rat and 176 & 67 bp for squirrel) were obtained. The detection limit of the assay was 0.1% meat in frankfurter formulation.